Methods and compositions for monitoring cell migration and identifying clinically relevant cytotoxic t lymphocyte activity

ABSTRACT

A three-dimensional reconstruct model containing solid layers of collagen and fibroblasts closely mimic the condition of patients in vivo, preserving in vivo phenotypic and functional characteristics of the cells. Reconstructs can be used to detect cellular migration, including active migration of cytotoxic T lymphocytes towards tumor cells, which can be measured by detecting tumor cell lysis. Reconstructs also can be used to identify factors that influence migration of cells, including chemokines that influence migration of cytotoxic T lymphocytes, as well as to identify tumor antigens

This application claims priority to co-pending provisional applicationSer. No. 60/324,907 filed Sep. 27, 2001, which is incorporated byreference in its entirety herein.

This invention was made with government funds from grants 1 P50 CA93372-01. The government has certain rights in the invention.

FIELD OF THE INVENTION

The invention relates to methods and compositions for monitoring cellmigration and identifying clinically relevant cytotoxic T lymphocyte(CTL) activity.

BACKGROUND OF THE INVENTION

A critical issue in cancer immunotherapy is raised by the finding thattumors grow in patients even when infiltrated with lymphocytes. Forexample, a brisk pattern of T cell infiltration into vertical growthphase melanoma is a powerful predictor of cure after surgery (Clark etal., 1989). Nevertheless, tumors frequently progress (Lee et al.,1999b). Furthermore, results from melanoma antigen-specific vaccinationssuggest vaccine effectiveness in eliciting T cell responses demonstrablein circulation and at the tumor site in the absence of clinicalresponses (Lee et al., 1999a; Finke et al., 1999; Nielson & Maricola,2000). An important related question is why lymphocytes that lyse tumortargets in vitro often do not demonstrate therapeutic effects afteradoptive transfer to patients in vivo (Maaser et al., 1999).

Multiple factors have been suggested to underlie the absence of CTLfunctions in vivo, such as (1) production of TGF-β by tumor cellsleading to impairment of CTL effector function, (2) downregulation ofHLA class I on tumor cells in vivo leading to absence of tumor lysis byCTL, (3) absence of co-stimulatory molecules on tumor cells leading toimpairment of CTL induction by direct tumor stimulation, (4) expressionof Fas ligand by tumor cells leading to inactivation of Fasreceptor-positive CTL, (5) emergence of tumor cell variants withepitope/antigen loss after tumor lysis by CTL in vivo, and (6)downregulation of ζ chain in CTL leading to loss of T cell function. Allof these factors have been identified in vitro, but it is unclear whichof them play a role in vivo. Elucidation of these questions wouldgreatly impact T-cell based adoptive and active cancer immunotherapies.

These questions cannot be addressed directly in patients. Studies on Tcell functions have been performed in tumors in situ using analyses ofRNA expression (Finke et al., 1999). However, these studies have usedbiopsy material containing a mixture of cells and have used an RNAamplification method. Thus, the cell type which is the source of RNA(stroma cells, fibroblasts, tumor cells, immune effector cells) cannotbe identified. Moreover, RNA amplification is non-linear and may bebiased.

Thus far, relevant in vitro models are lacking. Traditionally, CTL areraised in two-dimensional mixed lymphocyte tumor cell culture (MLTC)that include either long-term cultured tumor cells to stimulate PBMC forCTL induction or disaggregated tumor tissue with tumor infiltratinglymphocytes (TIL), with both cultures grown directly on plasticsurfaces. However, a study in melanoma patients has shown thatcharacterization of CTL responses using tumor cell lines does notreflect the in vivo exerted anti-tumoral activity (Friedl et al., 1998).For example, melanoma-reactive human CTL derived from skin biopsies ofdelayed type hypersensitivity reactions, when stimulated with autologouslong-term cultured tumor cells in vitro, demonstrated increased skewingof the Vβ T cell receptor (TCR) repertoire with increasing time inculture (Rao et al., 2000). This suggests the selection of a CTLpopulation in culture that does not reflect the composition of Tlymphocytes in vivo. In agreement with that study, TCR expressed by CTLraised in MLTC are not expressed in situ (Crowston et al., 1997).Changes in TCR repertoire of CTL following in vitro culture indicatechanges in antigen recognition. Thus, CTL in situ may recognizedifferent antigens than CTL derived from long-term cultures.

In addition to the alterations of T cells in MLTC upon culture, the useof long term cultured tumor cells in MLTC is disadvantageous. Forexample, colorectal carcinoma (CRC) cells often lose HLA class Iexpression in culture, whereas the same tumors express these moleculesin situ (Schroder, 1995). Similarly, the tumor suppressor gene p16 isexpressed in ˜70% of primary CRC, but in only ˜10% of CRC cell lines(Luger & Schwarz, 1990). Furthermore, irradiated tumor cells often usedin MLTC may not produce all of the factors that non-irradiated tumorcells produce; these factors may affect the induction or effector phaseof CTL.

Chemokines play a major role in inducing migration of lymphocytes,neutrophils, monocytes, macrophages and dendritic cells to the tumorsite. Chemokines may be incorporated into vaccines to increase vaccineefficacy at the site of the antigen-presenting cell or attached to ananti-tumor antibody to attract the effector cells to the tumor site.Alternatively, tumors may be transduced with chemokines to attracteffector cells or CTL may be transduced with chemokine receptors(Biragyn & Kwak, 2000).

Thus, there is a need in the art for model systems that can be used toidentify clinically relevant behaviors of CTL, including tumor celllysis and active migration, as well as systems that can be used toidentify chemokines that influence active migration of CTL towards tumorcells.

BRIEF SUMMARY OF THE INVENTION

One embodiment of the invention provides a composition of matter fordetecting active migration of cells. The composition comprises (a) afirst solid layer comprising collagen and fibroblasts, (b) a cellularlayer in contact with the first solid layer and comprising a first celltype, (c) a second solid layer covering the cellular layer andcomprising collagen and fibroblasts, and (d) a third solid layer incontact with the second solid layer and comprising collagen,fibroblasts, and a second cell type. The second cell type is a migratorycell. In a preferred embodiment, the first cell type is a tumor cell andthe second cell type is a cytotoxic T lymphocyte.

Another embodiment of the invention provides a method of preparing acomposition of matter. Collagen and fibroblasts are deposited in avessel to form a first solid layer comprising collagen and fibroblasts.Cells of a first cell type are deposited on the first solid layer toform a cellular layer in contact with the first solid layer. Collagenand fibroblasts are deposited on the cellular layer to form a secondsolid layer comprising collagen and fibroblasts. The second solid layercovers the cellular layer. Collagen, fibroblasts, and cells of a secondcell type are deposited on the second solid layer to form a third solidlayer in contact with the second solid layer. The third solid layercomprises collagen, fibroblasts, and cells of the second cell type.

Even another embodiment of the invention provides a method of detectingactive migration of migratory cells. First positions of migratory cellsin the third solid layer of a composition are detected at a first time.Second positions of the migratory cells are detected at a second time.The compositions comprises (a) a first solid layer comprising collagenand fibroblasts, (b) a cellular layer in contact with the first solidlayer and comprising a first cell type, (c) a second solid layercovering the cellular layer and comprising collagen and fibroblasts, and(d) a third solid layer in contact with the second solid layer andcomprising collagen, fibroblasts, and the migratory cells. A differencein the first and second positions indicates active migration of themigratory cells. In a preferred embodiment, the first cell type is atumor cell and the second cell type is a cytotoxic T lymphocyte.

Another embodiment of the invention provides a method of detectingactive migration of cytotoxic T lymphocytes. A composition comprising(a) a first solid layer comprising collagen and fibroblasts, (b) acellular layer in contact with the first solid layer and comprisingtumor cells, (c) a second solid layer covering the cellular layer andcomprising collagen and fibroblasts, and (d) a third solid layer incontact with the second solid layer and comprising collagen, fibroblastsis inspected to determine whether cytotoxic T lymphocytes are present inthe second solid layer.

Yet another embodiment of the invention provides a method of detectingactive migration of cytotoxic T lymphocytes towards tumor cells. Tumorcell lysis is detected in a first composition and a second composition.The first composition comprises (1) a first solid layer comprisingcollagen and fibroblasts, (2) a cellular layer in contact with the firstsolid layer and comprising tumor cells, (3) a second solid layercovering the cellular layer and comprising collagen and fibroblasts, and(4) a third solid layer in contact with the second solid layer andcomprising collagen, fibroblasts, and cytotoxic T lymphocytes. Thesecond composition comprises (1) a first solid layer comprising collagenand fibroblasts, (2) a cellular layer in contact with the first solidlayer and comprising tumor cells, (3) a second solid layer covering thecellular layer and comprising collagen and fibroblasts, and (4) a thirdsolid layer in contact with the second solid layer and comprisingcollagen, fibroblasts, and control lymphocytes that are not cytotoxic Tlymphocytes. Increased tumor cell lysis in the first compositioncompared with the second composition indicates active migration of thecytotoxic T lymphocytes towards the tumor cells in the firstcomposition.

A further embodiment of the invention provides a method of detectingcytotoxicity of cytotoxic T lymphocytes. A first percentage of killedtumor cells in a first composition is compared with a second percentageof killed tumor cells in a second composition. The first compositioncomprises (a) a first solid layer comprising collagen and fibroblasts,(b) a cellular layer in contact with the first solid layer andcomprising tumor cells, (c) a second solid layer covering the cellularlayer and comprising collagen and fibroblasts, and (d) a third solidlayer in contact with the second solid layer and comprising collagen,fibroblasts, and cytotoxic T lymphocytes. The second compositioncomprises (a) a first solid layer comprising collagen and fibroblasts,(b) a cellular layer in contact with the first solid layer andcomprising tumor cells, (c) a second solid layer covering the cellularlayer and comprising collagen and fibroblasts, and (d) a third solidlayer in contact with the second solid layer and comprising collagen,fibroblasts, and phytohemagglutinin-treated blast cells. An increasebetween the first and the second percentages indicates cytotoxic Tlymphocyte-induced killing of tumor cells in the first composition.

Another embodiment of the invention provides a method of identifying atumor antigen that binds to a T cell receptor of a cytotoxic Tlymphocyte that induces killing of tumor cells. A clone of cytotoxic Tlymphocytes that induces killing of tumor cells is identified bycomparing a first percentage of killed tumor cells in a firstcomposition with a second percentage of killed tumor cells in a secondcomposition. The first composition comprises (a) a first solid layercomprising collagen and fibroblasts, (b) a cellular layer in contactwith the first solid layer and comprising tumor cells, (c) a secondsolid layer covering the cellular layer and comprising collagen andfibroblasts, and (d) a third solid layer in contact with the secondsolid layer and comprising collagen, fibroblasts, and cytotoxic Tlymphocytes. The second composition comprises (a) a first solid layercomprising collagen and fibroblasts, (b) a cellular layer in contactwith the first solid layer and comprising tumor cells, (c) a secondsolid layer covering the cellular layer and comprising collagen andfibroblasts, and (d) a third solid layer in contact with the secondsolid layer and comprising collagen, fibroblasts, andphytohemagglutinin-treated blast cells. An increase between the firstand the second percentages indicates cytotoxic T lymphocyte-inducedkilling of tumor cells in the first composition. Host cells that expressan HLA restriction element autologous for the cytotoxic T lymphocytesand comprising cDNA of the tumor cells are tested to identify a hostcell that induces cytokine release in cytotoxic T lymphocytes of theclone. Tumor cell cDNA is isolated from the host cell. The tumor cellcDNA encodes a tumor antigen. The invention also provides tumor antigensidentified using such methods.

Even another embodiment of the invention provides a method ofidentifying a chemokine that influences active migration of migratorycells. A chemokine receptor expressed by a first population of migratorycells is identified. The first population is contacted with a moleculethat specifically binds to the chemokine receptor. Active migration ofthe first population with active migration of a second population ofmigratory cells that has not been contacted with the first molecule. Adifference in active migration between the first and second populationsidentifies the chemokine as influencing active migration of themigratory cells. Active migration of the first and second populations isdetected by a method comprising the steps of: (1) detecting at a firsttime first positions of migratory cells in a third solid layer of acomposition comprising (i) a first solid layer comprising collagen andfibroblasts, (ii) a tumor cell layer in contact with the first solidlayer, (iii) a second solid layer covering the tumor cell layer andcomprising collagen and fibroblasts; and (iv) the third solid layer incontact with the second solid layer and comprising collagen,fibroblasts, and cytotoxic T lymphocytes; and (2) detecting at a secondtime second positions of the migratory cells, wherein a difference inthe first and second positions indicates active migration of themigratory cells.

Still another embodiment of the invention provides a method ofidentifying a chemokine or a cytokine that influences active migrationof migratory cells. Active migration of a first population of migratorycells is compared with active migration of a second population ofmigratory cells. A difference in active migration between the first andsecond populations identifies the chemokine or the cytokine asinfluencing active migration of the migratory cells. Active migration ofthe first population is detected by a method comprising the steps of:(a) detecting at a first time first positions of the first population ina third solid layer of a first composition, wherein the firstcomposition comprises (1) a first solid layer comprising collagen andfibroblasts, (2) a target cell layer in contact with the first solidlayer, (3) a second solid layer covering the target cell layer andcomprising collagen and fibroblasts; and (4) the third solid layer incontact with the second solid layer and comprising collagen,fibroblasts, and the first population, wherein the first compositionfurther comprises a molecule that specifically binds to a chemokine or acytokine; and (b) detecting at a second time second positions of thefirst population, wherein a difference in the first and second positionsindicates active migration of the first population. Active migration ofthe second population is detected by a method comprising the steps of:(a) detecting at a first time first positions of the second populationin a third solid layer of a second composition, wherein the secondcomposition comprises (1) a first solid layer comprising collagen andfibroblasts, (2) a target cell layer in contact with the first solidlayer, (3) a second solid layer covering the target cell layer andcomprising collagen and fibroblasts; and (4) the third solid layer incontact with the second solid layer and comprising collagen,fibroblasts, and the second population; and (b) detecting at a secondtime second positions of the second population, wherein a difference inthe first and second positions indicates active migration of the secondpopulation.

Yet another embodiment of the invention provides a method of identifyinga chemokine that influences active migration of cytotoxic T lymphocytestowards tumor cells. Tumor cell lysis is detected in a first compositionand a second composition. The first composition comprises (1) a firstsolid layer comprising collagen and fibroblasts, (2) a cellular layer incontact with the first solid layer and comprising tumor cells, (3) asecond solid layer covering the cellular layer and comprising collagenand fibroblasts, and (4) a third solid layer in contact with the secondsolid layer and comprising collagen, fibroblasts, and a first populationof cytotoxic T lymphocytes comprising a blocking molecule that preventsbinding of the chemokine receptor to its cognate chemokine. The secondcomposition comprises (1) a first solid layer comprising collagen andfibroblasts, (2) a cellular layer in contact with the first solid layerand comprising tumor cells, (3) a second solid layer covering thecellular layer and comprising collagen and fibroblasts, and (4) a thirdsolid layer in contact with the second solid layer and comprisingcollagen, fibroblasts, and a second population of cytotoxic Tlymphocytes, wherein the chemokine receptor of the second population isavailable to bind to its cognate ligand. The chemokine is identified asaffecting migration of cytotoxic T lymphocytes if tumor cell lysis isgreater in the second composition than the first composition.

Even another embodiment of the invention provides a method ofidentifying a cytokine that influences active migration of cytotoxic Tlymphocytes towards tumor cells. Tumor cell lysis is detected in a firstcomposition and a second composition. The first composition comprises(1) a first solid layer comprising collagen and fibroblasts, (2) acellular layer in contact with the first solid layer and comprisingtumor cells, (3) a second solid layer covering the cellular layer andcomprising collagen and fibroblasts, and (4) a third solid layer incontact with the second solid layer and comprising collagen,fibroblasts, and a first population of cytotoxic T lymphocytes, whereinthe first composition further comprises a blocking molecule thatprevents binding of a cytokine to its cognate receptor. The secondcomposition comprises (1) a first solid layer comprising collagen andfibroblasts, (2) a cellular layer in contact with the first solid layerand comprising tumor cells, (3) a second solid layer covering thecellular layer and comprising collagen and fibroblasts, and (4) a thirdsolid layer in contact with the second solid layer and comprisingcollagen, fibroblasts, and a second population of cytotoxic Tlymphocytes, wherein cytokines in the second composition are availableto bind to their cognate receptors. The cytokine is identified asaffecting migration of cytotoxic T lymphocytes if tumor cell lysis isgreater in the second composition than the first composition.

The invention thus provides methods and compositions for detectingactive migration of cells, particularly CTL, and identifying clinicallyrelevant behaviors of CTL.

DETAILED DESCRIPTION OF THE INVENTION

We have developed novel human organotypic culture systems, termed“reconstructs,” that closely mimic the conditions in patients in vivo,so that the in vivo phenotypic and functional characteristics of thecells are preserved as much as possible. In the three-dimensionalreconstruct, tissues such as human tumors, particularly melanoma andcolorectal carcinoma (CRC), are reconstituted in vitro using a mixtureof collagen and fibroblasts (lattices or matrices).

Activated fibroblasts in reconstructs play an important role in theactivation of T cells. The fibroblasts produce T cell survival factors(Murakami & Okada, 1997) and fibronectin, the latter stimulatingpredominantly resting lymphocytes (Somasundarum et al., 2000). Inaddition, fibroblasts produce various cytokines, growth factors andchemokines (Jacobs et al., 1998; Jakic-Razumovic et al., 1995; Gunzer etal., 2000). CRC-derived factors may exert inhibitory (e.g., TGF-β orIL-10) or stimulatory (e.g., IL-1 and GM-CSF) effects on T cellinduction. Some of the factors produced by fibroblasts and CRC cells mayaffect the induction, as well as the effector phase of T lymphocytes inthe reconstruct. Very little is known about the factors that affect theeffector phases of CTL because the relevant assays (e.g., ⁵¹Cr-releaseassay) do not allow the determination of these effects.

Lymphocytes bind to fibroblasts via lymphocyte function-associated Ag-1(LFA-1), intercellular adhesion molecule-1 (ICAM-1) and CD44. Theadhesive interaction stimulates fibroblasts to secrete inflammatorycytokines such as IL-1 and IL-6 (Biragyn & Kwak, 2000). The mainbiological role of IL-1 is the stimulation of Th cells to express IL-2receptors and secrete IL-2 (Herlyn et al., 2000). In addition, IL-1 canpromote adhesion of T cells by upregulation of adhesion molecules suchas ICAM-1, VCAM (vascular cell adhesion molecule)-1 and ELAM(endothelial leukocyte adhesion molecule). In the presence of IL-2 andIL-6, IL-1 induces the differentiation of T cells into cytotoxic T cells(Herlyn et al., 2000).

By investigating CTL functions in reconstructs, new combinationtherapies (e.g., with cytokines and/or chemokines) that may augment thecytolytic function of CTL in patients can be developed. For example,examination of CTL induction in the reconstruct can be used to identifynovel factors, not previously identified in traditional mixed lymphocytetumor culture (MLTC), which may be useful for enhancing the vaccineeffect of CTL-derived antigens. Furthermore, CTL that are raised againstfresh tumor tissues may recognize different, clinically more relevantantigens than the CTL raised in traditional MLTC, in which tumor cellsand lymphocytes are cocultured directly on plastic surfaces.Identification of the most effective CTL directed against fresh tissuesin a reconstruct model may lead to more effective adoptive CTL andvaccination immunotherapies as compared to conventional strategies basedon MLTC-derived CTL.

Reconstruct models have several advantages over conventional in vitroassays (4 or 18 hr ⁵¹Cr-release assay) and in vivo/in situ assays (11)(see Table 1). The cytolytic functions of T cells are usually tested invitro in ⁵¹Cr release assay. However this assay does not mimic the invivo situation. First, it is of short duration and therefore may notallow detection of antigenic modulation of tumor cells, including tumorantigens and HLA, as well as studies on the effect of cytokines producedby tumor cells on CTL effector function. Second, it includes singletumor cell suspensions and not tumor tissues, thus not allowingdetection of eventual negative effects of the growing tumor on T cellfunction. Third, the ⁵¹Cr-release assay uses artificially high E:T cellratios. Fourth, the assay does not take into account T cell migrationinto tumor tissues. Furthermore, studies of T cell kinetics, e.g. thekinetics of direct tumor cell lysis by CTL over time can only be studiedin the reconstruct model. This model is also advantageous for studies onthe mechanism of tumor cell lysis by CTL with the ultimate goal ofenhancing CTL function in adoptive immunotherapies of cancer patients.TABLE 1 Advantages of reconstruct models for characterizing CTL activityAssay systems ⁵¹Cr release In vivo/in Parameter (CTL) assay Reconstructsitu assays Tumor or T no yes yes cell/stroma interaction T cell kineticno yes no Time period of T 4-18 hr 4 weeks indefinitely cell/tumor cellinteraction T cell migration no yes no Mechanism of biased (in vivo yeslimited tumor killing by factors neglected) T cells Manipulation of Tbiased (in vivo yes limited and/or tumor factors neglected) cellsRequired CTL to high (>20) low (<1) unknown tumor cell ratioDetermination of no yes no effect of cytokines and chemokines on T cellfunctionReconstructs and Their preparation

Compositions of the invention (“reconstructs”) comprise four layers. Onelayer (the “first solid layer”) comprises collagen and fibroblasts. A“cellular layer” is in contact with the first solid layer and comprisesa first cell type, also termed herein a “target cell.” A “second solidlayer” covers the cellular layer and comprises collagen and fibroblasts.Finally, a “third solid layer” is in contact with the second solidlayer. The third solid layer comprises collagen, fibroblasts, and asecond cell type.

Typically, neutralized type I collagen is used in layers of areconstruct. Fibroblasts can be obtained from a variety of sources,including primary tissues (e.g. human neonatal foreskin) and fibroblastcell lines derived from neonatal human foreskin (e.g., FF2443).

In preferred embodiments, the first cell type is a tumor cell, such as acell of a colon tumor, biliary tumor, brain tumor (e.g., glioblastoma,medulloblastoma), breast tumor, cervical tumor, choriocarcinoma,endometrial tumor, esophageal tumor, gastric tumor, hematological cancer(e.g., acute lymphocytic and myelogenous leukemia, multiple myeloma,AIDS associated leukemias, adult T-cell leukemia, lymphoma),intraepithelial tumor, liver tumor, lung tumor, neuroblastoma, ovariantumor, pancreatic tumor, prostate tumor, rectal tumor, sarcoma(including leiomyosarcoma, rhabdomyosarcoma, liposarcoma, fibrosarcomaand osteosarcoma), skin tumor (including melanoma), testicular tumor,renal tumor, bladder carcinoma, or choriocarcinoma. Either primary tumorcells or tumor cell lines can be used. For example, suitable colon tumorcell lines are described in Example 5.

The second cell type typically is a migratory cell, such as a leukocyte(e.g., a neutrophil, macrophage, dendritic cell, or monocyte) orlymphocyte (e.g., a natural killer cell or lymphocyte, particularly acytotoxic T lymphocyte (CTL).

Reconstructs can be prepared as follows. Briefly, collagen andfibroblasts are deposited in a vessel to form the first solid layer.Wells of a 24-well plastic tissue culture plate provide a convenientvessel, although other plastic or glass vessels can be used. Targetcells are deposited on the first solid layer to form the cellular layer.The second solid layer, which covers the cellular layer, is formed bydepositing collagen and fibroblasts on the cellular layer. Finally,collagen, fibroblasts, and a second cell type are deposited on thesecond solid layer to form the third solid layer. Methods of preparingreconstructs are described in detail in the specific examples, below.

Methods of Detective Active Migration

Compositions of the invention can be used to detect active migration ofcells, such as leukocytes (e.g., neutrophils, macrophages, or monocytes)or lymphocytes (e.g., natural killer cells or lymphocytes, particularlyCTLs). Positions of migratory cells are detected at a first time in thethird solid layer of a composition of the invention. Positions of themigratory cells are detected at a second time. A difference in the firstand second positions indicates active migration of the migratory cells.For example, in one embodiment, human dermis is recapitulated in vitrousing mixture of collagen and fibroblasts (lattices or matrices).Melanoma cells can be included in the reconstruct. Melanoma cellmigration through collagen matrix, which is dependent on the interactionof α2β1 integrin (expressed by melanoma cells with collagen (Maaser etal., 1999), can be detected.

One or more compositions can be used to detect a difference in thepositions of migratory cells. For example, compositions can be fixed andsectioned, and the cells can be stained by conventional histologicaltechniques, as described in the specific examples, below.

Use of Reconstruct Models to Identify Factors that Modulate CTL Activityand Migration

Reconstructs of the invention can be used to identify factors thatactivate or inhibit CTL activity against tumor cells in vivo or whichmodulate CTL migration. The identification of these factors can greatlyimpact adoptive immunotherapy with CTL and active immunotherapy withCTL-defined antigens. For example, factors that enhance CTL inductionand/or effector function in the reconstruct can be added to adoptive andactive immunotherapies. Factors that impair CTL induction and/oreffector function can be functionally inactivated by CTL manipulation exvivo (adoptive CTL transfer) and/or tumor modulation in vivo (genetherapy).

The effector lytic mechanisms of CTL are usually tested in 6 hr orovernight ⁵¹Cr-release assays. Chromium-release assays areunsatisfactory for identifying relevant factors, however, because thetumor cells are in contact with the CTL for too short a time andfibroblast are absent. In contrast, the importance of fibroblast- andtumor-derived factors for CTL activity can easily be evaluated inreconstructs. In fact, additional CTL and/or tumor markers should beexpressed in a reconstruct because fibroblasts produce various cytokinesand growth factors and because collagen stimulates T cells (18). This issupported by the fact that the minimally required E:T ratio to inducetumor lysis by CTL is much lower in a reconstruct than in an MLTC.

Factors that potentially play a role in the activity of CTL in areconstruct model include, but are not limited to, CD markers (e.g.,CD3, CD4, CD8, CD25/IL2-Rα, CD45, CD45RO, CD28), adhesion molecules(e.g., E-cadherin, CD44, CD54/ICAM 1/My13, CD11a/LFA 1,CD106/VACAM/51-10C9, CD29/β1 integrin for VCAM, β3 integrin), HLA (e.g.,class I, class II, β2 microglobulin/BM63), co-stimulatory molecules(e.g., CD80/B7-1, CD86/B7-2) tumor antigens (e.g., MAGE-1, tyrosinase,MART/MELAN A, p91A, MUM-1, p21^(ras) with a point mutation at position12, p210 product of bcr-abl rearrangements, HER-2/neu, p53, RB, BAGE,GAGE, SV40 T antigen, human papillomavirus E6 and E7 gene products,Epstein-Barr virus EBNA-1 gene product), apoptosis/necrosis molecules(e.g., FAS, FAS-L, granzyme B, perforin, TNF-α), cytokines (e.g.,GM-CSF, TFN, IFNγ, IL-1, IL-2, IL-4, IL-6, IL-8, IL-10, TNF-α),chemokines and their receptors (e.g., IP-10, MIP-1α, CCR5/MCP-1α, RANTESreceptor, RANTES, CCR1/MCP-1 receptor, CCR-2/MCP-1 receptor, CXCR1/IL-8receptor; for a complete list, see Homey et al., 2002), and growthfactors (e.g., TGFβ).

Reconstruct models described herein are particularly useful foridentifying the role of individual cytokines and chemokines in CTLmigration. For example, cytokines that are secreted by fibroblasts ortumor cells and that modulate CTL induction or effector function againsttumor cells can be identified. Cytokines that directly potentiate CTLeffector function are largely unknown. Other cytokines may directlyinduce target cell necrosis, such as TNF and IFNγ.

In one embodiment, reconstructs are formed with and without theinclusion of fibroblasts. If omission of fibroblasts in suchreconstructs affects CTL effector function, techniques such asmicroarray analysis and IHC can be used to identify which cytokines areexpressed by fibroblasts isolated from the reconstruct. Antibodiespenetrate through the collagen/fibroblast matrix of a reconstruct. Thus,identified cytokines can be inactivated using neutralizing antibodies,and the effect of such inactivation on the migration of CTL toward tumorcells can be determined in a reconstruct model. The identified cytokinescan be used in conjunction with adoptive CTL immunotherapy or asadjuvants with vaccines of CTL-defined antigens.

Reconstructs also are unique in their ability to permit investigation ofthe role of chemokines and their receptors in the migration of CTLtowards tumor cells. Chemokines control leukocyte and activated T cellmigration to sites of inflammation and immune reaction. Fibroblastspresent in a reconstruct may be stimulated by IL-1 (produced by tumorand/or T cells) to produce chemokines (e.g., MIP-1α, MCP-1, RANTES) thatmay stimulate CTL migration. However, because fibroblasts typically areevenly dispersed throughout all layers of a reconstruct, they will notnecessarily provide a chemokine gradient. In contrast, tumor-derivedchemokines can provide a gradient, which will attract the T cells andcause T cell migration from a distant layer to the tumor layer,resulting in tumor apoptosis.

Thus, reconstructs can be used to identify the chemokines and/orchemokine receptors that are involved in the migration of CTL. Chemokinereceptors expressed by CTL can be identified, for example, usingantibodies. Antibodies can be obtained from commercial sources (e.g.,R&D Systems, Inc., Minneapolis, Minn.) or can be raised using standardtechnologies. Alternatively, RT-PCR using known primers can be used.

Recognizing that one receptor can bind several chemokines, an ELISAassay can be used to determine whether tumor cells secrete acorresponding chemokine into serum-free medium.

Blocking molecules, such as chemokine antagonists or antibodies, can beused to block the identified receptor to test the effect of blockingbinding between an identified chemokine and its receptor. In oneembodiment, CTL are incubated with an antibody (e.g at 0.5 μg/ml finalconcentration) before they are placed in a reconstruct. Inhibition ofCTL migration relative to isotype-matched control antibody at the sameconcentration can be determined by measuring inhibition of tumor cellapoptosis. If tumor cell apoptosis is greater in the controlreconstruct, the chemokine is identified as involved in active CTLmigration toward the tumor cells.

To confirm the role of the chemokine in CTL migration, the chemokineitself can be blocked using chemokine-specific antibodies or othermolecules that bind to the chemokine and prevent its binding to itsreceptor. To further confirm the role of the identified chemokine in CTLmigration and induction of tumor cell apoptosis, the chemokine can beadded to a layer of tumor cells in a reconstruct (e.g., at 1-10 μg/ml),which should increase both CTL migration toward the tumor cells andtumor cell apoptosis. Finally, if the identified chemokine enhances CTLmigration, transfection of the identified chemokine into tumor cells tooverexpress the chemokine will lead to enhanced tumor cell apoptosisinduced by CTL. Transfection can be achieved, for example, using avector, such as an adenovirus vector, that expresses the chemokine.

Relevant cytokines or chemokines identified in a reconstruct model can,for example, be fused to a tumor-specific antibody to attract adoptivelytransferred CTL or can be incorporated into vaccines to activate T cells(Biragyn & Kwak, 2000). Alternatively, a tumor can be transduced withone or more chemokines or cytokines in vivo or CTL may be transducedwith chemokine or cytokine receptors.

Methods of Detecting Cytotoxicity of CTLs

Cytotoxicity of CTLs against target cells (e.g., tumor cells) in thecellular layer of a reconstruct can be detected by any means known inthe art, including biochemical and morphological methods. See thespecific examples, below. For example, apoptotic tumor cells can bedetected in a TUNEL assay or by microscopic examination.

In one embodiment, pairs of reconstructs comprise either CTL orphytohemagglutinin (PHA)-treated blast cells, preferably obtained fromthe same individual. The percentage of killed tumor cells are detectedand compared between the two reconstructs. An increase between thepercentage of killed cells in the reconstruct comprising CTL and thepercentage of killed cells in the reconstruct comprising the controlcells indicates cytotoxicity of the CTLs against the tumor cells.Alternatively, the percentages of viable cells in the two cellularlayers can be compared. In another embodiment, the percentage of killedtumor cells (or remaining viable cells) in a series of reconstructs isdetected over various times.

Methods of Identifying Tumor Antigens

Effective CTLs identified using reconstructs of the invention can beused to identify tumor antigens. Tumor antigens are antigens that areexpressed either only on tumor cells but not on normal cells or areexpressed are a greater level on tumor cells compared with normal cells.Preferably, tumor antigens stimulate T cell responses. Examples of suchtumor antigens include products of random point mutations in cellulargenes (e.g. p91A, MUM-1), oncogene products (e.g., p21^(ras) with apoint mutation at position 12, p210 product of bcr-abl rearrangements,HER-2/neu), mutated tumor-suppressor gene products (e.g., p53, RB),products of silent genes not normally expressed in most tissues (e.g.,MAGE 1, -3, BAGE, GAGE), viral gene products in virus-associatedmalignancies (e.g., SV40 T antigen, human papillomavirus E6 and E7 geneproducts, Epstein-Barr virus EBNA-1 gene product), and products oftissue-specific genes expressed in the tissue from which a tumor isderived (e.g., tyrosinase, p100, MART-1). Tumor antigens that stimulateCTL responses can be used in the preparation of therapeuticcompositions.

CTL antigens usually are recognized by HLA class I-restricted CD8⁺ orCD4⁺ CTL (Somasundarum et al., 2000; Jacob et al., 1997; townsend etal., 1986; Yasukawa et al., 1989; hayashi et al., 1992; Go et al., 1993)and less frequently by HLA non-restricted CD8⁺ CTL (Band et al., 1989).In one embodiment, cDNA encoding the autologous HLA restriction elementand a library of autologous tumor cell cDNA library is co-transfectedinto host cells (e.g., COS cells). Pools of cDNA are tested forreactivity with CTLs, for example in a cytokine release assay. ReactivecDNA clones are isolated, and the encoded antigen is characterized.Details are provided in the specific examples, below.

A peptide isolation method also can be used to identify tumor antigens,as is well known in the art. See Weber, 2002.

All patents, patent applications, and references cited in thisdisclosure are incorporated by reference in their entireties.

While the invention has been described with respect to specific examplesincluding presently preferred modes of carrying out the invention, thoseskilled in the art will appreciate that there are numerous variationsand permutations of the above described systems and techniques that fallwithin the spirit and scope of the invention as set forth in theappended claims.

EXAMPLE 1

Identification of CTL Against Human Melanoma Cells

Melanoma patient 793 had a primary vertical growth phase (VGP) tumorwhich was excised in 1983 and has been in remission since. PMBC obtainedfrom patient 793 in 1989 were stimulated with autologous melanoma cells793 (from day 0 on) and IL-2 (from day 7 on). Three CD4⁺ clones withcytolytic activity against autologous tumor cells were isolated, ofwhich one (clone A) was studied in detail. CTL793 clone A lysed theautologous melanoma cells WM793, but not the metastatic variant cells1205Lu derived from serial passages of WM793 cells both in vitro and invivo in nude mice (Juhasz et al., 1993), although the variant cellsexpress the same HLA as the parental cells.

There was no significant lysis of autologous Epstein Barr virus (EBV)-Bcells or NK- or LAK-sensitive target cells. These findings were furtherconfirmed in cytokine (IFN-γ) release assays. Of the various allogeneicmelanoma cells used as targets in CTL assays, only three that share HLAB57[17] with the autologous WM793 cells were consistently andsignificantly lysed and lysis was blocked by anti-B57[17] MAb (as wellas by anti-CD3, -CD4 and -class I MAbs).

To confirm HLA B57[17] dependency of target cell lysis, HLA B57 wascloned from WM793 cells and transfected into allogeneic, HLA non-matchedWM1158 melanoma cells. Transfected, but not non-transfected, WM1158cells were significantly lysed by CTL 793 clone A.

Thus, the CD4⁺ CTL 793 clone A described here clearly interacts with HLAclass I molecules in the lysis of melanoma cells. Characterization ofCTL clone 793A is shown in Table 2. TABLE 2 Characteristics of melanomacells and anti-melanoma CTL Cell line/clone CD4⁺ 793 CD8⁺ 793 MarkerWM793 CTL clone A CTL HLA (% pos. cells) HLA class I >95 >95 >95 HLAclass II >90 74 >95 CD markers (% pos. cells) CD3 − >99 >90 CD4 NA >99<1 CD8 NA <1 >98 Co-stimulatory molecules (% pos. cells) B7-1 7 6 NTB7-2 3 4 NT CD40 3 14 NT CD40-L 6 24 NT Adhesion molecules (% pos.cells) β1 integrin 100 60 NT CD44 92 97 NT CD54 (ICAM) >95 <20 NTCytokines (RIA/ELISA/FACS) IFNγ − + + TNFα − + + GM-CSF − + + TGFβ + − −IL-1 + − − IL-2 − − − IL-4 − − − IL-8 + − NT IL-10 − − NT Apoptosismarkers (% pos. cells) FAS >95 >95 NT FASL 10 <5 NT Chemokines MCP-1 +NT NT Rantes NT NT NT MIP-1α − NT NT Chemokine receptors CCR1 NT + NTCCR2 NT + NT CCR5 NT + NTNT = not tested;NA = not applicable.

EXAMPLE 2

Generation of CD8⁺ CTL

CD8⁺ CTL were generated from the PBMC of primary melanoma patient 793 inan MLTC, and IL-2 was added to the cultures as early as day 3. Thephenotype of this CTL population is predominantly (>80%) CD8⁺. The CD8⁺CTL were reactive in ⁵¹Cr-release and/or cytokine release assays againstallogeneic melanoma cells matched with autologous melanoma cells atHLA-A1, A29, or B57[17] loci. Thus, the uncloned CTL may recognizeseveral antigens/epitopes in association with different HLA. There wasno significant reactivity of the CD8⁺ CTL with NK, LAK or autologous andnine different allogeneic EBV-B targets. The phenotypic and functionalmarkers of the CD8⁺ CTL 793 are summarized in Table 3. TABLE 3 Lysis ofvarious HLA-matched and -unmatched target cells and IFN-γ secretion byCD8⁺ 793 CTL IFN-γ Target cell % Maximal secretion Designation OriginHLA subtype^(a) cytoxicity^(b) (U/ml)^(c) WM793 autologous A1, A29,B57[17], 40.1^(d) 10^(d) primary melanoma B35, DRB1 11, DQB1 0301 DM196allogeneic A23(9), Aw34, B57[17], N.D.^(e)  2.2^(d) metastatic melanomaB44, DR1, DR3, DRw1, DRw52 ME9874 allogeneic A2, A24, B57[17],  8.3^(d) 5.9^(d) metastatic melanoma B60, Cw3, Cw7, DR7, DQ2 A375 allogeneic A1,A2, B57[17], —, N.D. 41.2^(d) metastatic melanoma C6, — 1205LU^(f)autologous A1, A29, B57[17],  6.6 <0.01 metastatic variant B35, DRB1 11,DQB1 0301 WM75 allogeneic A2, A29, B12w44, 24.2^(d)  4.6^(d) metastaticmelanoma DR4, DR7 WM98 allogeneic A1, A3, B8, DR3  8.1 <0.01 metastaticmelanoma WM164 allogeneic A24, —, B7, —, C7, —, N.D. <0.01 metastaticmelanoma DR13, —, DQ1, DQ6, DRw52 WM1158 allogeneic A11, A24, B16,B60(40), C3, —, 45^(d) <0.01 metastatic melanoma DR13, DR4, DQ3, DQ6,DRw52, DRw53 793 EBV-B autologous B cells A1, A29, B57[17],  0  1.3 B35,DRB1 11, DQB1 0301 888EBV-B allogeneic B cells A1, A24, B52, B55, N.D.<0.01 C1, C7, DR15, — 1363EBV-B allogeneic B cells A1, A2, B44, B51, C1,N.D. <0.01 —, DR1, — 1088EBV-B allogeneic B cells A1, A2, B8, B44, C5,N.D. <0.01 —, DR4, DR17 1102EBV-B allogeneic B cells A2, A24, B55, B62,C3, —, DR4, DR15 N.D. <0.01 4226EBV-B allogeneic B cells A24, A32, B27,B38, C3, —, DR4, DR15 N.D. <0.01 K562 NK-sensitive N.D.  0 <0.01erythroleukemia cells Daudi lymphokine-activated N.D.  0 <0.01 killercell-sensitive lymphoma cells^(a)Based on tumor cell typing, except for WM75, WM98, DM196, DM198,ME9874. HLA types of WM793 cells and those matching between WM793 andother cells are in bold.^(b)CTL culture supernatants obtained after 24 hr of stimulation withvarious stimulator cells and IFNγ were tested for cytokine production byRIA.^(c)Lysis of labeled tumor target cells was determined in ⁵¹Cr releaseassay.^(d)p < 0.05 when compared to corresponding cpm values of spontaneousrelease of ⁵¹Cr by target cells and/or spontaneous release of cytokineby CTL.^(e)ND, not determined.^(f)Metastatic variant cells 1205LU were derived from primary WM793cells by serial passage of these cells both in vitro and in vivo in nudemice (Juhasz et al., 1993).

EXAMPLE 3

CD4⁺ CTL 3122 Derived from Lymphocytes Infiltrating a Melanoma SpleenMetastasis

CD4⁺ CTL were generated from the lymphocytes infiltrating spleenmetastases of a melanoma patient by repeated stimulation of the CTL withmetastatic melanoma cell line WM3122 and IL-2. When tested forcytotoxicity in a ⁵¹Cr-release assay, the short-term CTL line (2-3months in culture) significantly lysed the autologous melanoma cells andnot allogeneic melanoma cells (WM793) or NK cell target (K562). Thephenotype of this cell line was predominantly CD4⁺ (>75% of the cells).

EXAMPLE 4

T Cells Derived from Melanoma-Infiltrated Lymph Nodes

Tumor-infiltrated lymph nodes from patients 3406, 3407, 3408, and 3409were used to generate CTL. Mechanically disaggregated cells wereincubated for 7 days, followed by repeated IL-2 stimulations every 7days. On day 28, T cells were restimulated with short-term culturedautologous melanoma cells and IL-2. These lymphocytes were both CD4⁺ andCD8⁺. TIL 3406 significantly lysed autologous melanoma cells in a⁵¹Cr-release assay.

EXAMPLE 5

Establishment of Primary Colorectal Carcinoma Cell Lines

Colorectal carcinoma cell lines were established by mincing the tissuein Leibovitz/MCDB medium supplemented with 5% fetal bovine serum, 400 mML-glutamine, 5.6% NaHCO₃, 5 mg/ml streptomycin, 10,000 U/ml penicillin,250 μg/mg fungizone, and 5 mg/ml insulin, and plating the cells in thesame medium in 24-well plates pre-coated with 1% gelatin, followed byincubation in a 5% CO₂ incubator. Medium of the cultures was changedweekly, and confluent cultures were subcultured with trypsin.

In most cases, the HLA phenotypes of patients, as determined incomplement-dependent cytotoxicity or by PCR using allele-specificprimers (HLA DR, DP, and DQ), were confirmed for autologous tumor cellsby FACS or PCR analyses using allele-specific monoclonal antibody (MAb)or primers, respectively. Some HLA types were expressed on the CRC linesonly after treatment of these cells with IFN-γ. All cell lines expressedthe CRC-associated antigen C017-1A/GA733 and all WC lines weretumorigenic in either nude or SCID mice. TABLE 4 Primary CRC cell linesCRC Tissue of Patients' HLA-types and HLA expression by tumor cells lineorigin A B Cw DR DP DQ WC007 rectum,  1, 3² 35 n.e.¹ DR², 1³, 4² n.e.n.e. primary WC008 rectum,  1², 3 57(17) n.e. DR², 7³ n.e. n.e. primaryWC010 rectum, 25³, 29³, 3³ 18³, 58³(17) 3³  7³, 9³ n.e. n.e. primaryWC011 colon, 19³  5 or 21 7³, 8³  2³, 11³(5) n.t.⁴ n.e. primary WC012colon,  2 14, 35 7³, 8³  1⁵ (0101/0102/0104), n.e. n.e. primary 14⁵(1401/1407) WC013 colon,  1, 32(25) 22³, 44(12) 3³, 4³  3, 13(6) n.e.n.e. primary WC016 colon,  2³, 3  5³, 41³ 6³, 7³ n.e. n.e. n.e. primary¹n.e. = not expressed on tumor cells.²Expression on tumor cells only upon IFN-γ treatment.³HLA types are derived from lymphocytes and tumor expression has not yetbeen verified.⁴n.t. = not tested.⁵Expression verified by PCR only.

EXAMPLE 6

Cytotoxicity of CTLs in Tumor Reconstruct Models

CD4⁺ CTL that were generated from the PBMC of primary melanoma patient793 in MLTC were tested for the ability to induce growth regression ofautologous melanoma cells in a human dermis reconstruct model. Humandermis reconstructs were made by mixing normal human skin fibroblastswith type I collagen, and collagen was allowed to constrict inDulbecco's modified Eagle's medium (DMEM) supplemented with 10% human ABserum. After 24 hr, WM793 cells (1×10⁵) were seeded on each contractedcollagen matrix, and the gel was allowed to attach and grow intumor/fibroblast (1:1) medium. After 24 hr, an equal number of CTL(1×10⁵) was mixed with normal fibroblasts and collagen gel and layeredon top of the collagen-tumor gel. Thus, CTLs need to migrate throughcollagen layer to reach the melanoma cells.

Collagen cultures were maintained for 4 more days until cell harvestingand/or fixation. In control experiments, CTL were replaced by equalnumbers of T cell blasts derived by stimulating 793 PBMC with 1% v/vPHA-M (Gibco) for 72 hr and further propagation in IL-2 containingmedium. Reconstructs were stained on day 7 with hematoxylin and eosin.

Cultures containing autologous PHA blasts contained large numbers ofhealthy WM793 tumor cells. In contrast, the dermis reconstruct cultureestablished with 793 CTL contained predominantly dead tumor cells. Inmany areas of the CTL reconstruct cultures, the melanoma cells hadcompletely disappeared, whereas a continuous layer of live melanomacells was seen in the control cultures. Notably, E:T ratio of 1 washighly effective in the reconstruct, while only marginally effective inMLTC (Somasundarum et al., 2000).

An apoptosis (TUNEL) assay demonstrated that experimental tumor cellcultures undergo significantly higher rates of apoptosis in thereconstruct as compared to control tumor cell cultures (p<0.05;Student's t-test). CD25 (IL-2 receptor) staining of the control culturesrevealed little non-specific staining (probably of dying PHA blasts),whereas there was strong staining of CTL 793. There was no significantstaining with isotype-matched control antibody.

RNA was isolated from CTL 793 grown for 5 days in the melanoma/dermisreconstruct. PCR analysis of cDNA using CD3 primers revealed that theRNA was derived from T cells.

EXAMPLE 7

Melanoma Dermis Reconstruct and Immunohistochemistry (IHC)

Normal human fibroblasts (FF2443) are isolated from the dermis ofneonatal human foreskins, cultured in DMEM with glutamine (Gibco/BRL,Gaithersburg, Md.), 8 mM HEPES (Sigma, St. Louis, Mo.), and 10% fetalbovine serum (FBS) (Hyclone, Logan, Utah). WM793 cells are cultured in 4parts MCDB153 (Sigma) and 1 part L15 medium (Gibco) supplemented with 2%FBS and 5 μg/ml insulin (Sigma). To determine the effect of IL-2 on CTLfunction in reconstruct, CTL are stimulated with autologous tumor cellsfor 4 days before incorporating them into the tumor (e.g., melanoma orcolorectal carcinoma) reconstructs. Control T cell blasts are derived bystimulating PBMC with T cell mitogen (1% v/v PHA-M, Gibco) for 72 hrfollowed by cultivation in IL-2 containing medium.

Normal human skin fibroblasts (FF2443) are added to neutralized type Icollagen (Organogenesis, Canton, Mass.); final concentration of 0.8-1mg/ml) in minimal essential medium (MEM) (BioWhittaker, Walkersville,Mass.), 1.66 mM L-glutamine (Gibco), 10% human AB serum and 0.21% sodiumbicarbonate (BioWhittaker). Three hundred-fifty μl offibroblast-containing collagen (1.4×10⁵ cells per ml) are added per wellof a 24-well tissue culture plate (Falcon, Becton Dickinson, FranklinLakes, N.J.). The mixtures are allowed to constrict in DMEM with 10%human AB serum.

After 24 hr, a total of 1×10⁵ WM793 cells (Hsu et al., 1999) are seededon each contracted collagen matrix and allowed to attach for 2 hr.Following incubation, the culture medium is exchanged by one part DMEMsupplemented with 10% human AB serum and mixed 1:1 with melanoma cellculture medium supplemented with 2% human AB serum. After 24 hr, 250 μlof type I collagen mixed with FF2443 (1.5×10⁵ cells per ml) and a totalof 1×10⁵ lymphocytes are added to the collagen/fibroblast/melanomacultures and allowed to constrict.

Cultures are maintained submerged in the medium supplemented with 2%human AB serum for 4 more days until cell harvesting and/or fixation.Reconstructs are fixed in 10% neutral-buffered formalin (FisherScientific, Pittsburgh, Pa.) for 4 hr at room temperature, stored in 70%ethanol at 4° C. and embedded in paraffin. For frozen sections, tissuesare dehydrated in increasing concentrations of sucrose solution,embedded in OCT medium, and frozen on dry ice, followed by liquidnitrogen freezing.

For histopathological evaluation, 6-8 μm frozen or paraffin-embeddedsections are stained with hematoxylin-eosin (H&E). IHC for humanproliferation marker Ki-67 (clone MIB-1, IgG1, Immunotech, Westbrook,Me.) and other markers is performed on serial sections using anavidin-biotin-peroxidase system kit (Vector Laboratories, Burlingame,Calif.) and 3,3′-diaminobenzidine tetrahydrochloride (Sigma) aschromogen. Antigens are retrieved by microwave heat treatment in citratebuffer (BioGenex, San Ramon, Calif.). Prior to incubation with thepositive control mouse antibody to human Ki-67 in a humidified chamberat room temperature for 1-2 hr, non-specific binding is blocked with 10%normal horse serum. Isotype-matched antibody is used as negative controlfor each staining.

Between each incubation step, slides are rinsed twice in PBS for 3-5min. Biotin-labeled horse anti-mouse secondary antibody is applied for30 min at room temperature, followed by incubation with a preformedavidin-biotinylated enzyme complex for 30 min. After color developmentby addition of the chromogen and counterstaining with Mayer'shematoxylin, sections are mounted and evaluated under a lightmicroscope. Cultured, pelleted CTL, melanoma cells, or fibroblasts areused as positive control cells, which are fixed or frozen according topreparation of the reconstruct tissues.

Blocking antibodies are used for function analyses at a concentration of10 and 2 μg/ml as described in (Somasundarum et al., 2000).Alternatively, viral chemokine and chemokine receptor homologs are usedas described in (Biragyn & Kwak, 2000; Staib et al., 2001; Juhasz etal., 1993).

EXAMPLE 8

Comparison of CTL Activity in Reconstructs and in MLTC

CTL induction with fresh tissues in reconstruct and isolation of CTL(and melanoma cells) from reconstruct followed by propagation of CTL inMLTC. Fresh lymphocyte-rich tumor involved lymph node or cutaneousmetastases specimens obtained within 3 hr of surgery are minced in coldRPMI 1640 (GIBCO) medium containing 100 IU of PennStrep (GIBCO). Finelyminced tissues (1 mm³) are mixed with 250 μl of collagen and layered onfreshly gelled fibroblast-collagen matrix. Cultures are submerged inreconstruct medium supplemented with complement-deactivated 10% human ABserum for 5-6 days. On day 7, reconstruct medium is replaced by T cellmedium (RPMI 1640+5% IL-2+10% human AB serum). On day 14, if there areno or too few tumor cells in the reconstruct cultures, T cells arere-stimulated with freshly defrosted, irradiated tumor tissues (1 mm³)using fresh T cell medium. If tumor cells are growing in culture on day14, T cells are cultured only with T cell medium. On day 21, cells aremicrodissected or isolated using antibody-coated beads, and analyzed byFACS using specific antibodies.

To further expand the already antigen-committed CTL, collagensupernatant containing T cells and a few non-adherent melanoma cells iscollected by separating it from plastic-adherent melanoma cells andcultured in plastic wells for 4 weeks with periodic stimulation of tumorcells (derived from the adherent cells) and IL 2. To obtain sufficientnumber of cells for specificity and HLA restriction analyses, T cellsare periodically stimulated with autologous tumor cells, EBV-B cells asAPC and IL-2. T cells are phenotyped and characterized for theircytotoxic activity against fresh (cryopreserved and defrosted) melanomacells, melanoma cells derived from the reconstruct, and cells derivedfrom melanoma cell lines established outside the reconstruct usingneutral red dye assay (Somasundarum et al., 2000).

EXAMPLE 9

Cloning of Selected Antigens Recognized by CTL that are Highly Effectivein a Reconstruct Model

HLA restriction element cloning. HLA restriction element cloning can beperformed as described in (Somasundarum et al., 2000). Primerscorresponding to the 5′ and 3′ end of the correct HLA gene are designedusing the information available at the IMGT/HLA sequence data base (URLhttp file type, www host server, ebi.ac.uk domain name,imgt/hla/directory). The primers are flanked by short sequences forrestriction enzyme recognition. First strand cDNA is synthesizedfollowing standard protocols and used for PCR reaction with Roche'sExpand High Fidelity PCR system. The use of the high fidelity PCR systemreduces the risk of artificially introduced mutations during the PCRreaction. After cleavage of the DNA with the correct restriction enzymesand agarose gel purification, the PCR fragment is cloned into anexpression plasmid such as pTracer™-CMV/BSD. This plasmid is useful,because the inserted GFP permits tracking of the transfection efficiencyin COS cells. HLA mismatched tumor target cells are transfected with theHLA clone, and lysis of the transfected target cells by the CTL isevaluated to test for the distribution of the antigen.

Tumor cell expression cDNA library construction and antigen cloning.Total RNA is isolated from tumor cells, and full length cDNAs aresynthesized using the SMART™ PCR cDNA synthesis kit, which allows highefficiency full length cDNA synthesis. After completion of the secondstrand synthesis, the DNA is polished and an adapter is attached. Afteradapter ligation and purification, the DNA is phosphorylated usingkinase. An expression plasmid (e.g. pcDNA3.1) is cut with the correctrestriction enzyme and dephosphorylated. Vector DNA and cDNA arecombined and ligated. The ligated DNA is transfected into a host cell(e.g., E. coli). Electroporation is preferred, although othertransfection methods, such as the CaCl₂ method, can be used.Alternatively, the cDNAs can be cloned into an expression phagemid(phagemid packaging and infection is more efficient thanelectroporation).

Twenty random clones are sequenced to determine the quality anddiversity of the library. Typically, approximately 250,000 independentclones are screened to find low abundance genes with 99% probability(Maniatis et al., 1989; Ausubel et al., 1993). Therefore the initiallibrary size preferably is at least 5×10⁵ independent clones. Cells,such as COS-7 cells (10⁴ per microtiter well), are co-transfected withthe HLA plasmid and the miniprep cDNA pools of 100 cDNA clones usingFuGene6 (Roche). Forty to 48 hr later the same number of T cells areadded to each well and incubated for 48 hr. Supernatants are be testedin cytokine release assay (Somasundarum et al., 2000). Wells with atleast 3 times the signal of control wells are considered positive. ThecDNA of putative positive wells is tested again in triplicate. Positivepools are subcloned until a single CTL-reactive cDNA clone is isolated.The clone is sequenced, and the sequence is compared with all availabledatabases, predominantly GenBank (URL http file type, www host server,ncbi.nlm.nih.gov domain name, Genbank/GenbankSearch.html directory).Computer prediction analysis can be used to help identify the CTLepitope, based on the HLA restriction element (see URL http file type,wehih.wehi.edu.au domain name, mhcpep/directory and URL http file type,www host server, brown.edu domain name,Research/TB-HIV_Lab/epimatrix.html directory).

Antigen characterization. The cDNA clone is transfected into autologousfibroblasts or EBV-B cells, and lysis of the transfected cells by theCTL is demonstrated to confirm the correct identity of the cDNA clone.The tissue distribution of the antigen can be determined by Northernblot analysis.

EXAMPLE 10

Characteristics of CTL007 and CTL007-F7 in MLTC and Reconstruct

CTL line 007 was established from patient 007 with rectal carcinomastage Dukes' B by stimulating PBMC with autologous CRC cells WC007,using IL-2 from day 7 on. CTL expressed CD3, CD4, CD25, HLA-DR, andTCRα/β, but not CD40 and CD40 ligand. CTL produced IL-2, IL-4, IL-10,IFN-γ, and TNF-α. Analysis of the cytotoxic activity of the CTL linerevealed lysis of autologous WC007 tumor cells in standard 4-6 h⁵¹Cr-release assays at E:T ratios as low as 10 (minimum E:T ratio of 10to cause significant tumor lysis of 12%; maximum lysis of 72% at E:Tratio of 50). Lysis of WC007 tumor cells was significant throughout the23 wk culture period of the CTL line, and lytic activity wassignificantly enhanced by treatment of the tumor cells with IFN-γ. TheCTL line also lysed allogeneic CRC cells HT-29, matched for HLA-A1 andWC016, matched for HLA-A3. Ag-specific lysis of WC007 CRC cells by theCTL line was significantly (p<0.05) inhibited by saturatingconcentrations of MAb W6/32 to HLA-class I, -A3, -DR, CD3, and CD4.Thus, lytic capacity of the uncloned CTL line is both HLA-class I (A1and A3)- and HLA-class II (DR)-dependent, and both CD3 and CD4 of theCTL are involved.

WC007 tumor cells were destroyed by CTL007 in the rectal carcinomareconstruct at an E:T ratio of 1. This ratio is ineffective in MLTC.Importantly, CTL were lytic in the reconstruct without the addition ofexogenous IL-2, whereas IL-2 was essential in MLTC. CTL migrated fromthe fibroblast/collagen layer into the tumor cell/collagen/fibroblastlayer which was placed on top of the CTL layer. In control cultures withPHA blasts, there was no significant lysis of tumor cells. Table 5 showsthat statistically significant lower total number of tumor cells andhigher number of apoptotic tumor cells (determined microscopically) arefound in reconstructs containing CTL versus reconstructs containing PHAblast controls. TABLE 5 Tumor-specific CTL007 decrease tumor cell growthand increase the number of apoptotic cells in a rectal carcinomareconstruct Total no. tumor cells % of apoptotic cells Lymphocyte (mean+/− SD/5 fields) (mean +/− SD/5 fields) CTL 007 41.2 +/− 7.04 11.8 +/−4.3 PHA blast 69.6 +/− 16.7  6.0 +/− 2.3 P value¹ 0.008 0.029¹Comparison of CTL 007 vs PHA blast values; Student's 2-sided t-test

CTL007 F7 was established similarly to CTL007. The CTL show stablegrowth for >3 months in culture. They are CD3 (99%) and CD4 (98%)positive, but CD8 negative. They lyse the autologous tumor cells(minimum E:T of 50 for significant lysis of 23%), but not autologousEBV-B cells, and allogeneic Daudi and K562 cells (<3.5% lysis). CTL007F7 lysed CRC WC007 cells in the reconstruct at E:T ratio of 5. Thisratio is ineffective in MLTC. Similar to the CTL007 reconstruct, CTL007F7 were lytic in the reconstruct in the absence of exogenous IL-2,whereas they were IL-2 dependent in MLTC. Control reconstructs with PHAblasts seemed to indicate crypt formation by the CRC cells and showed noevidence of tumor cell lysis. Control T cell blasts are derived bystimulating PBMC with T cell mitogen (1% v/v PHA-M, Gibco) for 72 hrfollowed by cultivation in IL-2 containing medium.

In a CTL reconstruct stained with anti-caspase antibody there was strongblack staining indicative of apoptotic cell death, whereas the samereconstruct stained with isotype-matched control antibody showed nosignificant staining. PHA blast control reconstruct stained withanti-caspase antibody showed very little evidence of apoptotic celldeath. Staining of CTL reconstruct for lymphocyte marker CD45 revealedspecific (red) staining of lymphocytes that migrated into the tumor cellarea. Control PHA blasts reconstructs showed fewer lymphocytes, most ofthem remaining outside the tumor area. Thus, CTL007 F7 was effective inthe reconstruct at a lower E:T ratio that was ineffective in MLTC.Notably, the results are indicative of CTL migration into the tumor celllayer, which was placed underneath the CTL layer. In contrast, in theexperiment with a different CTL (CTL007), the CTL migrated upwards intothe tumor cell layer. Thus, T cells seem to be able to migrate in bothdirections.

Table 6 shows significant lower total number of tumor cells and highernumber of apoptotic tumor cells in the CTL007 F7 reconstruct versus thePHA blast control reconstruct. TABLE 6 Tumor specific CTL007 F7 decreasetumor cell growth and increase the number of apoptotic cells in therectal carcinoma reconstruct Total number of tumor cells % of apoptoticcells mean ± SD/field mean ± SD/field Lymphocyte (8 fields) (8 fields)CTL007 F7 65.8 ± 12.9 21.6 ± 2.4 PHA blast 106.6 ± 30.3  10.1 ± 3.2 Pvalue¹ 0.003 <0.001¹Comparison of CTL007 F7 versus PHA blast values; Student's 2-sidedt-test.

EXAMPLE 11

Preparation of Reconstructs Containing Primary Melanoma Cells

Melanoma tissues are obtained within 6 hr after surgery. Tumors arewashed in RPMI 1640 medium containing 300 IU/ml penicillin, 300 μg/mlstreptomycin, 300 μg/ml amikacin, 50 μg/ml gentamycin, and 2.5 μg/mlfungizone, followed by washing in versene containing 100 μg/mlgentamycin (antibiotics from Gibco), and minced to obtain 0.5-1 mm³pieces. Reconstructs are prepared by mixing neutralized type I collagen(Organogenesis, Canton, Mass., final concentration: 0.8-1 mg/ml) in MEM(BioWhittaker, Walkersville, Md.) supplemented with 10% human AB serum(Gemini Bio-Products, Woodlands, Calif.), 1.66 mM L-glutamine, 0.2%NaHCO₃ with the tumor pieces. The collagen-tumor mixture is plated intowells of 24-well plates (Corning, 5-8 pieces/well/1 ml). After gelformation, 1 ml medium (tumor/T cell medium 1:1) is added followed byincubation at 37° C. T cell medium contains RPMI 1640 supplemented with216 mg/l L-glutamine, 50 μg/ml gentamycin 10 mM HEPES, 5×10⁻⁵ M2-mercaptoethanol (Sigma), and 10% heat-inactivated human AB serum(Gemini Bio-Products, Woodlands, Calif.). After 5 days incubation,medium is supplemented with 2% human natural IL-2 (AdvancedBiotechnologies, Columbia, Md.). The amount of IL-2 is graduallyincreased to 5% by week 3. If tumor cells start dying, T cells areisolated and placed into new reconstructs with defrosted tumor piecesfor T cell restimulation.

T cells and tumor cells are isolated for characterization by FACSanalysis using anti-CD3 and anti-Melan A antibody (124) coated Dynabeads (Dynal), respectively. For IHC analysis, reconstructs are fixed in10% neutral-buffered formalin (Fisher Scientific, Pittsburgh, Pa.) for 4hr at room temperature, stored in 70% ethanol at 4° C. and embedded inparaffin. For frozen sections, tissues are dehydrated in increasingconcentrations of sucrose solution, embedded in OCT medium and frozen ondry ice, followed by liquid nitrogen freezing. TABLE 7 No. vials eachwith ˜2 × 10⁶ cryopreserved Patient Established cell lines Reconstruct/lymphocytes Phenotype No. Tumor Fibroblasts EBV-B MLTC MLTC Reconstruct(MLTC) 3406 + + + both 5 0 CD4/CD8 3407 + + + MLTC 2 0 CD4/CD83408 + + + MLTC 2 0 CD4/CD8 3409 − − + both 1 0 CD4 3413 − − + both 5 0CD4 3443 + − + both 0 0 not tested 3445 + − + both 5 0 not tested 3450 +− + both 8 1 not tested 3451 + − + both 9 6 not tested 3453 + + + both 31 not tested 3454 − + − both 2 8 not tested 3456 − + + both 1 1 nottested 3457 − + + both 0 0 not tested 3458 + − − both 0 0 not tested3461 − − − both 0 0 not tested 3463 − − − both 0 0 not tested

EXAMPLE 12

Isolation of CTL from Fresh CRC Tissues in MLTC

Fresh tumor specimen obtained within 6 hr of surgery is minced, washed2× in cold RPMI 1640 medium containing antibiotics (penicillin [300IU/ml], streptomycin [300 μg/ml], amikacin [300 μg/ml] and fungizone[2.5 μg/ml]). An aliquot of minced tumor tissue is frozen in freezingmedia (90% human AB serum+10% DMSO) for future restimulation of MLTC.Minced tumor tissues (0.5-1 mm³ pieces) are cultured in RPMI 1640supplemented with 10% human AB serum in 24 well tissue culture treatedplates (Corning, Corning, N.Y.). On day 3, cultures are supplementedwith 5% purified human IL-2 (Advanced Biotechnologies, Columbia, Md.).On day 7, culture medium is replaced by RPMI 1640 supplemented with 10%human AB serum and 5% human IL-2 (T cell medium). On day 14, cultureswith growing T cells are transferred into a new well and cultured in Tcell medium. On day 21, T cell cultures are restimulated with irradiatedfrozen tumor tissues (3-4 0.5-1 mm³ pieces/well). This procedure isrepeated every 14 days or when growth of T cells slows down. Growing Tcells are harvested, frozen in liquid N2 and further characterized.Wells with predominant adherent tumor cell monolayer growth are washedand supplemented with tumor cell media (MCDB201 medium containing 20%L15, 2% FBS, 10 mM glutamine, 0.05% NaHCO₃, 2 μg/ml insulin, 5 ng/mlEGF, 2 μg/ml transferrin). Culture medium is replaced by fresh tumorcell medium every 7 days. Growing tumor cells are further characterizedas we have previously described in detail (Jacob et al., 1997).

EXAMPLE 13

Characterization of CTL Phenotype and Function

A ⁵¹Cr-release assay is used to determine tumor cell lysis by the CTL.If fresh (frozen and defrosted) tumor cells show high ⁵¹Cr release, theneutral red dye exclusion assay is used (Somasundarum et al., 2000). Theminimal E:T ratio needed for tumor cell lysis by the CTL derived fromthe reconstruct or MLTC can be compared.

For IHC evaluation of the reconstructs, 6-8 μm frozen orparaffin-embedded sections are stained with H&E. IHC for humanproliferation marker Ki-67 (clone MIB-1, IgG1, Immunotech, Westbrook,Me.), T cell markers CD4, CD8 and CD45, and tumor cell marker Melan-A orGA733 (45) is performed on serial sections using anavidin-biotin-peroxidase system kit (Vector Laboratories, Burlingame,Calif.) and 3,3′-diaminobenzidine tetrahydrochloride (Sigma) aschromogen. Ki-67 Ag is retrieved by microwave heat treatment in citratebuffer (BioGenex, San Ramon, Calif.). Non-specific binding is blockedwith 10% normal horse serum before incubation with the mouse antibodiesto these markers in a humidified chamber at room temperature for 1-2 hr.Isotype-matched murine anti-influenza virus antibody is used as negativecontrol for each staining. Between each incubation step, slides arerinsed twice in PBS for 3-5 min.

Biotin-labeled horse anti-mouse secondary antibody is applied for 30 minat room temperature, followed by incubation with a preformedavidin-biotinylated enzyme complex for 30 min. After color developmentby addition of the chromogen and counterstaining with Mayer'shematoxylin, sections are mounted and evaluated under a lightmicroscope. Cultured, pelleted T cells (PHA blasts), CRC cells, orfibroblasts are used as positive control cell lines, which will be fixedor frozen according to preparation of the reconstruct tissues. Lymphnodes (T cells), skin (fibroblasts), and CRC tissues are used aspositive control tissues. An In Situ Cell Death Detection kit (TUNELassay, Roche Molecular Biochemicals, Mannheim, Germany) is used forimmunohistochemical detection and quantification of apoptosis at singlecell level. Alternatively, sections can be stained with purified rabbitanti-human caspase 3 antibody specific for the active form of the enzyme(R&D Systems, Minneapolis, Minn.) after blocking sections with avidin-,biotin-, and protein-blocking agents (from Vector Laboratories, andCoulter-Immunotech) and rabbit antibody.

EXAMPLE 14

In Vitro Identification of Chemokines that Affect Active Migration ofCTL Towards CRC Cells In Vivo

CRC reconstructs with isolated CRC and CTL. A collagen-fibroblast layeris prepared by mixing neutralized type I collagen (Organogenesis,Canton, Mass., final concentration: 0.8-1 mg/ml) in MEM supplementedwith 10% FBS, 1.66 mM L-glutamine, 0.2% NaHCO₃ with allogeneic humanfetal colon fibroblasts (between passages 5-8, 1.8×10⁵/well, 5×10⁵/ml).Allogeneic fibroblasts will not provide alloantigenic stimulant toalready committed CTL. Four-hundred fifty μl of the mixture is platedinto wells of 24-well plates (Falcon, Becton Dickinson, Franklin Lakes,N.J.). After gel formation, 1 ml DMEM supplemented with 10% FBS and 50μg/ml gentamycin is added, followed by incubation at 37° C. One daylater, the medium is removed, and 10⁵ tumor cells are added on top ofthe first layer in 20 μl of medium. Tumor cells are allowed to attachfor 2 hr at 37° C., followed by adding 1 ml medium (50% DMEM, 50% tumorcell medium—MCDB 201 containing 20% L-15, 2% FCS, 10 mM L-glutamine,0.057% NaHCO₃, 2 μg/ml insulin, 5 ng/ml EGF, 2 μg/ml transferring. Toprovide a barrier between tumor cells and CTL, a fibroblast-collagenoverlay is added for 24 hr.

The following day, a fibroblast-collagen overlay containingtumor-specific CTL pre-incubated with a chemokine-specific antibody isprepared by mixing 4×10⁵/ml fibroblasts and 5×10⁵/well CTL with collagenmatrix, and 250 μl of the mixture are added/well. CTL incubated with anisotype-matched control antibody are used in control reconstructs.Cultures are incubated in medium (⅓ DMEM, ⅓ tumor cell medium, ⅓ T cellmedium containing RPMI 1640 supplemented with 216 mg/L L-glutamine, 50μg/ml gentamycin, 10 mM HEPES (Sigma), 5×10⁻⁵ M 2-mercaptoethanol, and10% heat-inactivated human AB serum) for four days.

Apoptotic cells are detected by in situ 3′-end-labeling of apoptoticfragmented DNA. An In Situ Cell Death Detection kit (TUNEL assay, RocheMolecular Biochemicals, Mannheim, Germany) can be used forimmunohistochemical detection and quantification of apoptosis at singlecell level. In another embodiment, apoptotic nuclei are detectedmicroscopically. Briefly, after rehydration, sections can be firstdigested with 1.25 μg/ml Proteinase-K for 30 minutes at 40° C. Sectionsare rinsed in 70%, 90%, and 95% ethyl alcohol and air-dried at roomtemperature, followed by incubation with a nucleotide-DNA polymerasecocktail containing biotin-labeled dCTP and dATP, and non-labeled dTTP,dGTP at a concentration of 0.01 mM, and 5 units/ml Klenow fragment ofDNA polymerase I (Invitrogen). A brown color can be developed using thestreptavidin-biotin complex (ABC) and 3-3′ diaminobenzidine substratefrom Vector Laboratories. Sections can be counterstained withhematoxylin. The amount of tumor cell apoptosis can be compared betweenblocked and unblocked reconstructs using a two-sample t-test. If tumorcell apoptosis is greater in the control reconstruct, the chemokine isidentified as involved in active CTL migration toward the tumor cells.

EXAMPLE 15

CTL Derived from the PBMC of a Colon Carcinoma Patient

PBMC from patient 013 (Dukes' stage C) were stimulated with irradiatedautologous, long-term cultured tumor cells in the presence of irradiatedautologous EBV-B cells. IL-2 (20 U/ml) was added to the cultures staringfrom the second week on. Stable CTL lines—C3 (CD8), D1 (CD4), E12 (CD8),A6 (CD4), F12 (CD8), and H7 (CD4)—were isolated that significantly lysedWC013 tumor cells (maximum lysis between 20 and >100% at E:T between12.5 and 50). No lysis of LAK or NK specific targets (Daudi cells orK562 cells, respectively) was observed. Four of the 5 CTL lines testedfor IFN-γ production produced significant amounts of the cytokine. Lysisof WC013 tumor cells by CTL E12 was HLA class I restricted and lysis ofCTL A6 was both HLA class I and II restricted.

EXAMPLE 16

Expression of TCR Vα Chains in CTLs of Reconstructs Containing MelanomaCells

TCR analysis of lymphocytes was performed in uncultured melanoma tissue,cells of MLTC, and reconstructs derived from patient 3451. Reconstructswere initiated by seeding minced tissue into bovine type I collagen inMEM supplemented with 10% human AB serum. Seven days later and thenweekly, cultures received RPMI 1640 supplemented with 10% human AB serumand 10% natural IL-2.

MLTC were initiated by seeding minced tissues in RPMI 1640 mediumsupplemented with 10% human AB serum into plastic culture dishes. Sevendays later and then weekly, cultures received 10% naturalIL-2-containing medium (RPMI 1640 plus 10% human AB serum).

Vα chains 1, 3, 5, and 10 were found in MLTC cultures. Vα chains 3 and 7were found in defrosted, uncultured tissue. Vα chain 10 was found indefrosted tissue cultured overnight. Vα chains 6, 7, 8, and 10 werefound in PHA blasts from defrosted tissue after 3 weeks in culture.

The TCR of T lymphocytes isolated from reconstructs are more closelyrelated to the TCR in situ than the TCR of T lymphocytes derived fromMLTC. Moreover, the TCR repertoire is much greater in T cells derivedfrom MLTC compared to those in a reconstruct, which suggests thatconsiderable TCR skewing occurs in MLTC. Similar TCR skewing occurredafter prolonged culture of the tissue in PHA.

EXAMPLE 17

Expression of TCR Vα Chains in Reconstructs Containing ColorectalCarcinoma Cells

Colorectal carcinoma tissues were cultured as described in the exampleabove. Vα chains 2, 6, 7, 8, 11, 13, 18, and 21 were found in thereconstructs. Vα chains 2, 3, 5, 6, 7, 11, and 12 were found inuncultured tissue. Vα chains 7, 12, 13, 17, and 21 were found in MLTCcultures.

As with the melanoma reconstructs described above, the TCR repertoire ofthe T lymphocytes derived from reconstructs were more similar to therepertoire of the lymphocytes in situ than the TCR repertoire of MLTClymphocytes.

EXAMPLE 18

CTL Migration Through Additional Collagen Layer and Tumor Cell Apoptosis

Collagen and fibroblasts were seeded on the bottom of a well. A tumorcell layer was seeded on top of the bottom layer. A collagen fibroblastlayer was seeded on top of this layer, followed by a very top layer ofcollagen, fibroblasts and T cells. Thus, T cells have to migrate througha collagen layer to reach tumor cells. The CTL killed the tumor cells asdetermined by apoptotic cell counts of H&E stained sections under themicroscope. The counts were performed by two different observers. Theapoptosis was significantly higher in CTL cultures than in controlcultures (no T cells or PHA blasts) (p<0.05, student's t test). SeeTable 8. TABLE 8 Apoptotic tumor cells in reconstructs with CTL007Reconstruct % of Significance Tumor Separating Apoptotic Total cellapoptotic (p value) of % values to cells T cells layer cells +/− SDnumber +/− SD cells +/− SD No T cells CTL PHA blast Observer #1 WC007 —100 ul  7.1 +/− 2.8 90.1 +/− 26.3 8.19 +/− 3.5 + +0.003 WC007 CTLoo7 100ul 23.5 +/− 8.7 80.5 +/− 38.8 31.7 +/− 11  +<0.0001 +<0.0001 WC007 PHAbl 100 ul 15.9 +/− 6.8 136.2 +/− 54.8  12.1 +/− 3.9 + + Observer #2WC007 — 100 ul 11.3 +/− 3.1 90.8 +/− 10.6 12.6 +/− 4.1 + −0.165 WC007CTLoo7 100 ul 33.3 +/− 6.8 88.7 +/− 11.9 37.5 +/− 6.7 +<0.0001 +<0.0001WC007 PHAbl 100 ul 16.5 +/− 6.2 104.8 +/− 13.3  14.5 +/− 4.4 − +

REFERENCES

-   Lee, K.-H., Wang, E., Nielsen, M.-B., Wunderlich, J., Migueles, S.,    Connors, M., Steinberg, S. M., Rosenberg, S. A., and    Marincola, F. M. 1999a. Increased vaccine-specific T cell frequency    after peptide-based vaccination correlates with increased    susceptibility to in vitro stimulation but does not lead to tumor    regression. J. Immunol. 163: 6292-6300.-   Clark, W. H., Jr., Elder, D. E., Guerry, D., IV, Braitman, L. E.,    Trock, B. J., Schultz, D., Synnestvedt, M., and Halper, A. C. 1989.    Model predicting survival in stage I melanoma based on tumor    progression. J. Natl. Cancer Inst. 81: 1893-1894.-   Lee, P. P., Yee, C., Savage, P. A., Fong, L., Brockstedt, D.,    Weber, J. S., Johnson, D., Swetter, S., Thompson, J., Greenberg, P.    D., Roederer, M., and Davis, M. M. 1999b. Characterization of    circulating T cells specific for tumor-associated antigen in    melanoma patients. Nat. Med. 6: 677-685.-   Finke, J., Ferrone, S., Frey, A., Mufson, A., and Ochoa, A. 1999.    Where have all the T cells gone? Mechanisms of immune evasion by    tumors. Immunol. Today 20: 158-160.-   Nielsen, M.-B. and Marincola, F. M. 2000. Melanoma vaccines: the    paradox of T cell activation without clinical response. Cancer    Chemother. Pharmacol. 46: S62-S66.-   Maaser, K., Wolf, K., Klein, C. E., Niggemann, B., Zänker, K. S.,    Brocker, E.-B., and Friedl, P. 1999. Functional hierarchy of    simultaneously expressed adhesion receptors: integrin alpha2beta1    but not CD44 mediated MV3 melanoma cell migration and matrix    reorganization within three-dimensional hyaluronan-containing    matrices. Mol. Biol. Cell 10: 3067-3079.-   Friedl, P., Entschladen, F., Conrad, C., Niggemann, B., and    Zänker, K. S. 1998. CD4⁺ T lymphocytes migrating in    three-dimensional collagen lattices lack focal adhesions and utilize    β1 integrin-independent strategies for polarization, interaction    with collagen fibers and locomotion. Eur. J. Immunol. 28: 2331-2343.-   Rao, W. H., Hales, J. M., and Camp, R. D. R. 2000. Potent    costimulation of effector T lymphocytes by human collagen type I. J.    Immunol. 165: 4935-4940.-   Crowston, J. G., Salmon, M., Khaw, P. T., and Akbar, A. N. 1997.    T-lymphocyte-fibroblast interactions. Biochem. Soc. Trans. 25:    529-531.-   Schroder, J. M. 1995. Cytokine networks in skin. J. Invest.    Dermatol. 105: 20S-24S.-   Luger, T. A. and Schwarz, T. 1990. Evidence for an epidermal    cytokine networks J. Invest. Dermatol. 95: 100S-104S.-   Murakami, S. and Okada, H. 1997. Lymphocyte-fibroblast interactions.    Crit. Rev. Oral Biol. Med. 8: 40-50.-   Somasundaram, R., Robbins, P., Moonka, D., Loh, E., Marincola, F.,    Patel, A., Guerry, D., and Herlyn, D. 2000. CD4⁺, HLA class    I-restricted, cytolytic T-lymphocyte clone against primary malignant    melanoma cells. Int J. Cancer 85: 253-259.-   Jacobs, N., Moutschen, M. P., Franzen-Detrooz, E., Boniver, V.,    Boniver, J., and Delvenne, P. 1998. Organotypic culture of    HPV-transformed keratinocytes: a model for testing lymphocyte    infiltration of (pre)neoplastic lesions of the uterine cervix.    Virchows Arch 432: 323-330.-   Jakic-Razumovic, J., Sale, G. E., Beauchamp, M. D., Storb, R., and    Sandmaier, B. M. 1995. CD8⁺ activated T lymphocytes produce an in    vitro skin graft-versus-host reaction in an organotypic skin culture    model. Transplantation 59: 69-78.-   Gunzer, M., Schälfer, A., Borgmann, S., Grabbe, S., Zänker, K. S.,    Brocker, E.-B., Kämpgen, E., and Friedl, P. 2000. Antigen    presentation in extracellular matrix: interactions of T cells with    dendritic cells are dynamic, short lived, and sequential. Immunity    13: 323-332.-   Biragyn, A. and Kwak, L. W. 2000. Designer cancer vaccines are still    in fashion. Nature Med. 6: 966-968.-   Herlyn, D., Wettendorff, M., Schmoll, E., Iliopoulos, D., Schedel,    I., Dreikhausen, U., Raab, R., Ross, A. h., jaksche, H., Scriba, M.,    and Koprowski, H. 1987. Anti-idiotype immunization of cancer    patients: modulation of the immune response. Proc. Natl. Acad. Sci.    USA 84: 8055-8059.-   Staib, L., Birebent, B., Somasundaram, R., Purev, E., Braumüller,    H., Leeser, C., Küttner, N., Li, W., Zhu, D., Wunner, W., Speicher,    D., Beger, H-G., Song, H., Diao, J., and Herlyn, D. 2001.    Immunogenicity of recombinant GA733-2E Ag (CO17-1A, EGP, KS1-4, KSA,    Ep-CAM) in gastrointestinal carcinoma patients. Int. J. Cancer, in    press.-   Juhasz, I., Albelda, S. M., Elder, D. E., Murphy, G. F., Adachi, K.,    Herlyn, D., Valyi-Nagy, I., and Herlyn, M. 1993. Growth and invasion    of human melanomas in human skin grafted to immunodeficient mice.    Am. J. Pathol. 143: 528-537.-   Hsu, M.-Y., Elder, D. E., and Herlyn, M. 1999. Melanoma: The Wistar    Institute (WM) cell lines. In: Human Cell Culture (Mas-   Maniatis, T., Fritsch, E. F., and Sambrook, J. 1989. Molecular    cloning: A laboratory manual. Cold Spring Harbor Laboratory.-   Jacob, L., Somasundaram, R., Smith, W., Monos, D., Basak, S.,    Marincola, F., Pereira, S., and Herlyn, D. 1997. Cytotoxic T-cell    clone against rectal carcinoma induced by stimulation of patient's    peripheral blood mononuclear cells with autologous cultured tumor    cells. Int. J. Cancer 71: 325-332.-   Townsend, A. R., Rothbard, J., Gotch, F. M., Bahadur, G., Wraith,    D., and McMichael, A. J. 1986. The eoptopes of influenza    nucleoprotein recognized by cytotoxic T lymphocytes can be defined    with short synthetic peptides. Cell 44: 959-968.-   Yasukawa, M., Inatsuki, A., and Kobayashi, Y. 1989. Differential in    vitro activation of CD4⁺CD8 and CD8⁺CD4. Herpes simplex    virus-specific human cytotoxic T cells. J. Immunol. 143: 2051-2057.-   Hayashi, Y., Hoon, D. S. B., Park, M. S., Terasaki, P. I.,    Foshag, L. J., and Morton, D. L. 1992. Induction of CD4⁺ cytotoxic T    cells by sensitization with allogeneic melanomas bearing shared or    cross-reactive HLA-A. Cell. Immunol. 139: 411-425.-   Go, C., Lancid, D. W., Fitch, F. W., and Miller, J. 1993. Anergized    T cell clones retain their cytolytic ability. J. Immunol. 150:    367-376.-   Barnd, D. L., Lan, M. S., Metzgar, R. S., and Finn. O. J. 1989.    Specific, major histocompatibility complex-unrestricted recognition    of tumor-associated mucins by human cytotoxic T cells. Proc. Natl.    Acad. Sci. USA 86: 7159-7163.-   Ausubel, F. M., Brent, R., Kingston, R. E., Moore, D. D.,    Seidman, J. G., Smith, J. A., and Struhl, K. 1993. Current protocols    in molecular biology.-   Jacob, L., Somasundaram, R., Smith, W., Monos, D., Basak, S.,    Marincola, F., Pereira, S., and Herlyn, D. 1997. Cytotoxic T cell    clone against rectal carcinoma induced by stimulation of a patient's    peripheral blood mononuclear cells with autologous cultured tumor    cells. Int. J. Cancer 71: 325-332.-   Weber, Peptide Vaccines for Cancer, Cancer Investigation 20,    208-221, 2002-   Homey et al., Chemokines: Agents for the Immunotherapy of Cancer?    Nature Rev. 2, 175, 2002

1. A composition of matter for detecting active migration of cells,comprising: (a) a first solid layer comprising collagen and fibroblasts;(b) a cellular layer in contact with the first solid layer andcomprising a first cell type; (c) a second solid layer covering thecellular layer and comprising collagen and fibroblasts; and (d) a thirdsolid layer in contact with the second solid layer and comprisingcollagen, fibroblasts, and a second cell type, wherein the second celltype is a migratory cell. 2-9. (canceled)
 10. A composition of matterfor detecting active migration of cytotoxic T lymphocytes, comprising:(a) a first solid layer comprising collagen and fibroblasts; (b) acellular layer in contact with the first solid layer and comprisingtumor cells; (c) a second solid layer covering the cellular layer andcomprising collagen and fibroblasts; and (d) a third solid layer incontact with the second solid layer and comprising collagen,fibroblasts, and cytotoxic T lymphocytes. 11-19. (canceled)
 20. A methodof detecting active migration of migratory cells, comprising the stepsof: (a) detecting at a first time first positions migratory cells in thethird solid layer of the composition of claim 1; and (b) detecting at asecond time second positions of the migratory cells, wherein adifference in the first and second positions indicates active migrationof the migratory cells. 21-28. (canceled)
 29. A method of detectingactive migration of cytotoxic T lymphocytes, comprising the steps of:(a) detecting at a first time first positions of cytotoxic T lymphocytesin the third solid layer of the composition of claim 10; and (b)detecting at a second time second positions of the cytotoxic Tlymphocytes, wherein a difference in the first and second positionsindicates active migration of the migratory cells.
 30. A method ofdetecting active migration of cytotoxic T lymphocytes towards tumorcells, comprising the steps of: (a) detecting tumor cell lysis in afirst composition comprising (1) a first solid layer comprising collagenand fibroblasts, (2) a cellular layer in contact with the first solidlayer and comprising tumor cells, (3) a second solid layer covering thecellular layer and comprising collagen and fibroblasts, and (4) a thirdsolid layer in contact with the second solid layer and comprisingcollagen, fibroblasts, and cytotoxic T lymphocytes; and (b) detectingtumor cell lysis in a second composition comprising (1) a first solidlayer comprising collagen and fibroblasts, (2) a cellular layer incontact with the first solid layer and comprising tumor cells, (3) asecond solid layer covering the cellular layer and comprising collagenand fibroblasts, and (4) a third solid layer in contact with the secondsolid layer and comprising collagen, fibroblasts, and controllymphocytes that are not cytotoxic T lymphocytes, wherein increasedtumor cell lysis in the first composition compared with the secondcomposition indicates active migration of the cytotoxic T lymphocytestowards the tumor cells in the first composition. 31-36. (canceled) 37.A method of detecting cytotoxicity of cytotoxic T lymphocytes,comprising the steps of: comparing a first percentage of killed tumorcells in a first composition comprising: (a) a first solid layercomprising collagen and fibroblasts; (b) a cellular layer in contactwith the first solid layer and comprising tumor cells; (c) a secondsolid layer covering the cellular layer and comprising collagen andfibroblasts; and (d) a third solid layer in contact with the secondsolid layer and comprising collagen, fibroblasts, and cytotoxic Tlymphocytes; with a second percentage of killed tumor cells in a secondcomposition comprising: (a) a first solid layer comprising collagen andfibroblasts; (b) a cellular layer in contact with the first solid layerand comprising tumor cells; (c) a second solid layer covering thecellular layer and comprising collagen and fibroblasts; and (d) a thirdsolid layer in contact with the second solid layer and comprisingcollagen, fibroblasts, and phytohemagglutinin-treated blast cells;wherein an increase between the first and the second percentagesindicates cytotoxic T lymphocyte-induced killing of tumor cells in thefirst composition. 38-41. (canceled)
 42. A method of identifying a tumorantigen that binds to a T cell receptor of a cytotoxic T lymphocyte thatinduces killing of tumor cells, comprising the steps of: (a) identifyinga clone of cytotoxic T lymphocytes that induce killing of tumor cells bya method comprising the steps of: (1) comparing a first percentage ofkilled tumor cells in a first composition comprising: (i) a first solidlayer comprising collagen and fibroblasts; (ii) a cellular layer incontact with the first solid layer and comprising tumor cells; (iii) asecond solid layer covering the cellular layer and comprising collagenand fibroblasts; and (iv) a third solid layer in contact with the secondsolid layer and comprising collagen, fibroblasts, and cytotoxic Tlymphocytes; with a second percentage of killed tumor cells in a secondcomposition comprising: (i) a first solid layer comprising collagen andfibroblasts; (ii) a cellular layer in contact with the first solid layerand comprising tumor cells; (iii) a second solid layer covering thecellular layer and comprising collagen and fibroblasts; and (iv) a thirdsolid layer in contact with the second solid layer and comprisingcollagen, fibroblasts, and phytohemagglutinin-treated blast cells;wherein an increase between the first and the second percentagesindicates cytotoxic T lymphocyte-induced killing of tumor cells in thefirst composition; (b) testing host cells that express an HLArestriction element autologous for the cytotoxic T lymphocytesidentified in step (a) and comprising cDNA of the tumor cells toidentify a host cell that induces cytokine release in cytotoxic Tlymphocytes of the clone; and (c) isolating tumor cell cDNA from thehost cell, wherein the tumor cell cDNA encodes a tumor-associatedantigen. 43-45. (canceled)
 46. Tumor-associated antigens identified bythe method of claim
 42. 47. A method of identifying a chemokine thatinfluences active migration of migratory cells, comprising the steps of:(a) identifying a chemokine receptor expressed by a first population ofmigratory cells; (b) contacting the first population with a moleculethat specifically binds to the chemokine receptor; and (c) comparingactive migration of the first population with active migration of asecond population of migratory cells that has not been contacted withthe first molecule, wherein a difference in active migration between thefirst and second populations identifies the chemokine as influencingactive migration of the migratory cells, and wherein active migration ofthe first and second populations is detected by a method comprising thesteps of: (1) detecting at a first time first positions of migratorycells in a third solid layer of a composition comprising (i) a firstsolid layer comprising collagen and fibroblasts, (ii) a tumor cell layerin contact with the first solid layer, (iii) a second solid layercovering the tumor cell layer and comprising collagen and fibroblasts;and (iv) the third solid layer in contact with the second solid layerand comprising collagen, fibroblasts, and cytotoxic T lymphocytes; and(2) detecting at a second time second positions of the migratory cells,wherein a difference in the first and second positions indicates activemigration of the migratory cells. 48-53. (canceled)
 54. A method ofidentifying a chemokine or a cytokine that influences active migrationof migratory cells, comprising the step of: comparing active migrationof a first population of migratory cells with active migration of asecond population of migratory cells, wherein a difference in activemigration between the first and second populations identifies thechemokine or the cytokine as influencing active migration of themigratory cells, and wherein active migration of the first population isdetected by a method comprising the steps of: (a) detecting at a firsttime first positions of the first population in a third solid layer of afirst composition, wherein the first composition comprises (1) a firstsolid layer comprising collagen and fibroblasts, (2) a target cell layerin contact with the first solid layer, (3) a second solid layer coveringthe target cell layer and comprising collagen and fibroblasts; and (4)the third solid layer in contact with the second solid layer andcomprising collagen, fibroblasts, and the first population, wherein thefirst composition further comprises a molecule that specifically bindsto a chemokine or a cytokine; and (b) detecting at a second time secondpositions of the first population, wherein a difference in the first andsecond positions indicates active migration of the first population, andwherein active migration of the second population is detected by amethod comprising the steps of: (a) detecting at a first time firstpositions of the second population in a third solid layer of a secondcomposition, wherein the second composition comprises (1) a first solidlayer comprising collagen and fibroblasts, (2) a target cell layer incontact with the first solid layer, (3) a second solid layer coveringthe target cell layer and comprising collagen and fibroblasts; and (4)the third solid layer in contact with the second solid layer andcomprising collagen, fibroblasts, and the second population; and (b)detecting at a second time second positions of the second population,wherein a difference in the first and second positions indicates activemigration of the second population. 55-65. (canceled)